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Article Direct Analysis of and in Urine Samples Using Single Drop Microextraction Technique In-Line with Capillary Electrophoresis

Anna Poliwoda * , Katarzyna Zieli ´nskaand Piotr P. Wieczorek

Faculty of Chemistry, University of Opole, Oleska 48, 45-042 Opole, Poland * Correspondence: [email protected]; Tel.: +48-77452-7116

 Academic Editors: Mihkel Koel and Marek Tobiszewski  Received: 20 February 2020; Accepted: 25 March 2020; Published: 29 March 2020

Abstract: The fully automated system of single drop microextraction coupled with capillary electrophoresis (SDME-CE) was developed for in-line preconcentration and determination of muscimol (MUS) and psilocin (PSC) from urine samples. Those two analytes are characteristic active metabolites of and Psilocybe , evoking visual and auditory hallucinations. Study analytes were selectively extracted from the donor (urine samples, pH 4) into the organic phase (a drop of layer), and re-extracted to the acidic acceptor (background electrolyte, BGE), consisting of 25 mM phosphate buffer (pH 3). The optimized conditions for the extraction procedure of a 200 µL urine sample allowed us to obtain more than a 170-fold enrichment effect. The calibration curves 1 were linear in the range of 0.05–50 mg L− , with the correlation coefficients from 0.9911 to 0.9992. The limit of detections was determined by spiking blank urine samples with appropriate standards, 1 1 i.e., 0.004 mg L− for PSC and 0.016 mg L− for MUS, respectively. The limits of quantification varied 1 1 from 0.014 mg L− for PSC and 0.045 mg L− for MUS. The developed method practically eliminated the sample clean-up step, which was limited only to simple dilution (1:1, v/v) and pH adjustment.

Keywords: psilocin; muscimol; single drop microextraction; capillary electrophoresis; urine; green chemistry

1. Introduction Some species of mushrooms (especially Amanita and Psilocybe) are sources of naturally occurring psychedelic substances. The best known hallucinogenic ingredients are (IBO), muscimol (MUS), (PSB), and psilocin. The main psychoactive constituents in Amanita species are ibotenic acid (IBO) and muscimol [1,2]. The psychoactive dose of muscimol is estimated to be in the range of 10–15 mg, whereas that of ibotenic acid is 50–100 mg [3]. Generally, 100 g of dried contains about 150 mg of MUS and 30 mg of IBO; therefore, 10 g of dried mushrooms can evoke a psychedelic effect. The toxicity of MUS–LD50 values in mice is 2.5 mg/kg [4]. Their psychotropic properties result from their ability to activate the N-methyl-d-aspartate glutamate and GABA receptors [5]. The components of Psilocybe are hallucinogenic . These mushrooms grow in Middle and North Europe, both Americas, Asia, Australia, and Oceania [2]. The common , , typically contains 1–2 mg psilocin/g dried mushroom. The hallucinogenic effect of psilocin is evoked at doses in the range from 8 to 10 mg [6]. According to the literature data, the LD50 value of PSC in mice is 290 mg/kg [7]. In contrast to MUS and IBO, these indole alkaloids have a similar chemical structure to the human , and behave as serotonin receptors [6,8,9].

Molecules 2020, 25, 1566; doi:10.3390/molecules25071566 www.mdpi.com/journal/molecules Molecules 2020, 25, 1566 2 of 12

The risks associated with the consumption of these mushrooms result not only from their toxicity, but also from the fact that they possess ambivalent potential. This means that they can evoke either favorable or adverse reactions to living organisms. Therefore, the measurement of those analytes, both in mushroom extracts and body fluids matrices, is essential in quantitative and qualitative analyses to investigate and monitor poisoning status. For this reason, it is significant to have a reliable and fast analytical method for diagnostic examination of intoxication with hallucinogenic mushrooms. At present, only a few analytical procedures exist, including the LC-MS/MS [10], GC-MS [11], or CE-MS [12] techniques, which were developed to determine muscimol and ibotenic acid in serum or urine samples. Generally, the available literature focuses upon the analysis of mushroom extracts in which the content of analyzed analytes is significantly higher [13–16]. A few more papers describe the methods of psilocybin and psilocin analysis in physiological fluids, the most popular of which are those using HPLC with a detector (MS) [17–23] and photodiode array detector (PDA) [24], or using electrochemical detection [9,25]. Some analytical procedures with GC-MS [26,27] and CE-UV [28] have also been used. However, a sample pretreatment step for sample clean-up and analyte preconcentration is often required. Consequently, in this work, we focused on two fungal hallucinogenic metabolites: muscimol and psilocin. These two compounds provide definite proof of hallucinogenic . To our knowledge, no analytical methods have been developed to simultaneously detect muscimol and psilocin in biological samples (urine). The method described in this work consists of a fully automated system, i.e., single drop microextraction technique in-line with capillary electrophoresis (SDME-CE) without any sample clean-up step. The proposed analytical procedure may be useful in forensic investigations. Recently, the combination of a single drop microextraction technique in-line with capillary electrophoresis was applied for the analysis of selected organic compounds [29–38]. The main advantage of the SDME-CE technique is that the simultaneous isolation, preconcentration, and determination of various analytes is possible in a fully automated test. In this case, the sample components (analytes) are selectively extracted into a thin layer of organic (extractant) that covers the drop of the acceptor phase suspended at the inlet end of the capillary. In the next step, analytes are re-extracted into the acceptor phase and analyzed in-line with a capillary electrophoresis method [39]. Due to the drop volume (a few nanoliters), the SDME technique is an analytical procedure that radically reduces the consumption of . Furthermore, high enrichment factors that result in low detection limits can be easily obtained.

2. Results and Discussion

2.1. Optimization of SDME-CE parameters For the optimization of the SDME-CE method, major factors, e.g., the volume of donor phase and its pH, the type of organic solvent used for drop formation, as well as the time of extraction, need to be investigated. Therefore, the first stage of this research included a procedure for optimizing the extraction conditions of aqueous solutions of standard substances (a mixture of MUS and PSC).

2.1.1. Selection of the Organic Phase and Drop Formation The applicability of SDME is limited to the appropriate choice of organic solvent to be used for drop formation. The applied solvent should have low and low in the aqueous donor and acceptor phases. A low density of organic solvent ensures the stability of a single drop. Additionally, it should have a good ability to extract analytes and high to form a well-settled phase. Therefore, nonpolar solvents (octanol, , , and ), as well as their mixtures (1:1, v/v) in which studied compounds are soluble, were tested. For example, when toluene was used separately, both were extracted with very low extraction efficiency (below 10 %). For comparison, dichloromethane and ethyl acetate could be used, but as an ingredient Molecules 20202020,, 2525,, 1566x FOR PEER REVIEW 3 of 12

of a mixture rather than as a single solvent, because of their . Usually, working with these of a mixture rather than as a single solvent, because of their volatility. Usually, working with these solvents causes problems with maintaining a stable drop hanging at the tip of the capillary. solvents causes problems with maintaining a stable drop hanging at the tip of the capillary. Finally, four studied solvent configurations (octanol, octanol with ethyl acetate, octanol with Finally, four studied solvent configurations (octanol, octanol with ethyl acetate, octanol with dichloromethane, and toluene with dichloromethane) allowed us to get the desired results (Figure 1). dichloromethane, and toluene with dichloromethane) allowed us to get the desired results (Figure1). However, the best enrichment factor was observed when octanol was applied; therefore, it was used However, the best enrichment factor was observed when octanol was applied; therefore, it was used in in further experiments. Moreover, octanol enabled us to obtain a stable organic surface (organic further experiments. Moreover, octanol enabled us to obtain a stable organic surface (organic phase) phase) that covered the drop of the acceptor phase suspended at the inlet end of the capillary. that covered the drop of the acceptor phase suspended at the inlet end of the capillary. Undesirable Undesirable phenomenon of drop dissolution with an extended extraction time were not present only phenomenon of drop dissolution with an extended extraction time were not present only with octanol. with octanol.

Figure 1. EffectEffect of organic solvent on the enrichment fa factorctor of the studied analytes in aqueous samples using the SDME-CE system. Sample concentration: 25 mg L−11, pH 4; CE conditions: BGE—25 mM using the SDME-CE system. Sample concentration: 25 mg L− , pH 4; CE conditions: BGE—25 mM phosphate bubufferffer pHpH 3;3; SampleSample volume:volume: 200 µµL;L; OrganicOrganic phase:phase: various organic solvents;solvents; extractionextraction time: 180 s; injection: 0.1 0.1 psi, psi, 1s; 1s; n = 6,6, RSD RSD << 6.2%.6.2%.

2.1.2. Adjustment of the Compo Compositionsition of Acceptor and Donor Phase The most crucial parameterparameter inin SDME-CESDME-CE isis thethe choicechoice ofof acceptoracceptor phase.phase. The acceptoracceptor phasephase aaffectffectss analyteanalyte extractionextraction during during single single drop drop microextraction microextraction procedures, procedure ass, wellas well as the as resolutionthe resolution and sensitivityand sensitivity of the of analytes the analytes during during CE separation. CE separation. Therefore, Therefore, the effect the of effect several of buseveralffers with buffers diff erentwith concentrationsdifferent concentrations and wasand tested.pHs was An tested. increase An of increase the pH ofof thethe acceptorpH of the phase acceptor (CE buphaseffer) (CE led tobuffer) high EOF,led to which high EOF, increased which the increased mobility the of mobility analytes, of butanalytes, also increased but also increased the deprotonation the deprotonation of the studied of the analytesstudied analytes and their and low their affinity low to affinity the organic to the phase organic (octanol). phase Furthermore,(octanol). Furthermore, under these under conditions, these problemsconditions, with problems peaks tailingswith peaks and tailings the Joule and heating the Joule effect heating arose. effect It was arose found. It thatwas 25foun mMd that phosphate 25 mM buphosphateffer (pH 3)buffer used as(pH a CE 3) electrolyte used as yieldeda CE bothelectrolyte efficient yielded and reproducible both efficient electrophoretic and reproducible resolution andelectrophoretic enabled to obtainresolution high an enrichmentd enabled to factors. obtain Furthermore, high enrichment the organicfactors. phaseFurthermore, volume the is a organic critical parameterphase volume for theis a extractioncritical parameter efficiency for and the kinetics. extraction By efficiency playing with and back-pressurekinetics. By playing and its with time, back the- influencepressure and of the its n-octanoltime, the volumeinfluence was of the investigated. n-octanol volume Although was the investigated. use of lower Although volumes ofthe organic use of extractivelower volumes phase of led organic to higher extractive extraction phase effi ledciency, to higher the repeatability extraction efficiency, values were the poor.repeatability values wereAnother poor. important parameter to obtain a high enrichment factor is the pH of the donor phase. In thisAnother case, the important donor phase parameter (aqueous to obtain samples) a high at di enrichmentfferent pH values factor (2;is the 3; 4; pH 6; 8;of 10) the was donor examined. phase. In this case, the donor phase (aqueous samples) at different pH values (2; 3; 4; 6; 8; 10) was examined.

Molecules 2020, 25, 1566 4 of 12

Molecules 2020, 25, x FOR PEER REVIEW 4 of 12 Figure2 demonstrates the e ffect of donor phase pH on the enrichment of MUS and PSC by the Figure 2 demonstrates the effect of donor phase pH on the enrichment of MUS and PSC by the SDME SDME in-line CE system. For both analytes, the best enrichment factor was obtained with an acidic in-line CE system. For both analytes, the best enrichment factor was obtained with an acidic pH. pH. Generally, the increase of donor phase pH from 4 to 10 resulted in decreasing the enrichment Generally, the increase of donor phase pH from 4 to 10 resulted in decreasing the enrichment factors factors of the studied analytes. Good recoveries were achieved at pH 4, so this was selected for of the studied analytes. Good recoveries were achieved at pH 4, so this was selected for further further experiments. experiments.

FigureFigure 2. 2.Preconcentration Preconcentration of MUS and and PSC PSC versus versus pH pH of ofthe the donor donor phase. phase. Sample Sample concentration: concentration: 25 mg L−1, a1t pH in range 2–8; CE conditions: BGE—25 mM phosphate buffer pH 3; Sample volume: 200 25 mg L− , at pH in range 2–8; CE conditions: BGE—25 mM phosphate buffer pH 3; Sample volume: 200µLµ; L;Organic Organic phase: phase: octanol; octanol; extraction extraction time: time: 180 180s; injection: s; injection: 0.1 psi, 0.1 1s; psi, n 1s;= 6,n RSD= 6, < RSD 4.8%.< 4.8%.

TheThe obtained obtained results results confirmedconfirmed thatthat inin thethe case of the muscimol and and psilocin, psilocin, the the acidic acidic pH pH of of thethe sample sample solution solution increased the the preconcentration preconcentration effect effect of the ofthe studied studied analytes analytes.. As for As samples for samples with withpH pH≤ 4, the4, obtained the obtained enrichment enrichment factor factorwas the was highest. the highest. This pH This influenced pH influenced the presence the presenceof analyte of ≤ analytemolecules molecules in aqueous in aqueous sample sample solution solution in a cationic in a cationic form (MUS form pKa (MUS 4.8 pKaand 8.4, 4.8 andPSC 8.4,pKa PSC 9.4). pKa At the 9.4). Atsame the sametime, time,the cationic the cationic form form of the of thestudied studied analytes analytes limited limited its itsdirect direct extraction extraction through through the the hydrophobichydrophobic octanol octanol phasephase thatthat coveredcovered the drop of an acceptor phase phase during during the the SDME SDME process. process. Therefore,Therefore, presumably presumably onon thethe donordonor side,side, therethere must be a process that, that, despite despite the the presence presence of of positivelypositively charged charged muscimol muscimol andand psilocinpsilocin molecules,molecules, facilitatesfacilitates their diffusion diffusion into into oct octanol.anol. This This factor is supposed to be orthophosphoric acid, which is present in the sample (as it was used for pH factor is supposed to be orthophosphoric acid, which is present in the sample (as it was used for adjustment). Thus, taking into account the phosphoric acid pKa values (2.12; 7.21; and 12.67), at pH pH adjustment). Thus, taking into account the phosphoric acid pKa values (2.12; 7.21; and 12.67), at 4, the dominant form in the sample solution is negative ion H2PO4− (almost 100% molecules). For pH 4, the dominant form in the sample solution is negative ion H2PO4− (almost 100% molecules). example, this negatively charged counter ion (H2PO4−) is able to interact (forming an ion-pair) with a For example, this negatively charged counter ion (H PO ) is able to interact (forming an ion-pair) highly hydrophilic, positively charge amino group in 2muscimol.4− This phenomenon seems to decrease with a highly hydrophilic, positively charge amino group in muscimol. This phenomenon seems to the overall muscimol hydrophilicity and increase the affinity of the neutral ion-pair for the decrease the overall muscimol hydrophilicity and increase the affinity of the neutral ion-pair for the hydrophobic phase of octanol. There is no direct proof of the existence of an ion pair between the hydrophobic phase of octanol. There is no direct proof of the existence of an ion pair between the studied analytes and phosphate ions. Presumably, by analogy of amino acids and separations studiedby reversed analytes-phase and liquid phosphate chromatography, ions. Presumably, the effect by analogyof anionic of aminoion-pairing acids reagent and peptide interaction separations also byinfluence reversed-phases the enrichment liquid chromatography, of the cationic form the of eff analytesect of anionic [40]. Furthermor ion-pairinge, reagentat the beginning interaction of the also influencesproject, 80% the w enrichment/w of thewas cationic used to form acidify of analytesthe samples [40]. (pKa Furthermore, 4.75). In this at thecase, beginning the desirable of the project,preconcentration 80% w/w acetic effect acidwas wasnot observed, used to acidify which themay samples indirectly (pKa confirm 4.75). the In presence this case, of the an desirableanionic ion-pairing reagent effect.

Molecules 2020, 25, 1566 5 of 12 preconcentration effect was not observed, which may indirectly confirm the presence of an anionic ion-pairing reagent effect. Furthermore, if the extraction proceeds as long as the concentration gradient is preserved, the enrichment factor will be 1, but only in the case when the equilibrium stage is observed, and the concentration of the form of studied analytes is the same in both the acceptor and donor phases. In the developed SDME method, the donor phase pH was 4, whereas acceptor phase pH was 3 (phosphate buffer). Such a small change in the pH value of the aqueous phases significantly influences the number of negatively charge H2PO4− ions presented in the solutions. In the acceptor phase (pH 3), the number of H2PO4− ions decreases (only about 75–80% of the total molecules, according to the dissociation diagram). Thus, fewer ion pairs are formed on the acceptor side, and therefore, an increase of positively charged analyte molecules is observed. Moreover, the presence of ionized particles prevents them from re-entering the organic phase of octanol. It is enough to observe concentration gradient (and thus the rate of mass transfer) of the diffusing species. Furthermore, on the basis of similarities with membrane extraction, the concentration difference, ∆C, over the organic phase, can be written as:

∆C = α C α C D D − A A where CD and CA are the total concentrations in the donor (sample) and acceptor phases, respectively, and αD and αA are the fractions of the analytes that are in extractable (ion-pair) form in the indicated phase [41,42]. In studied SDME system, the αD and αA values seem to play a significant role; therefore, the enrichment of the studied analytes, observed at an acceptor phase of pH 3, was sufficient for the present purposes. The effect of donor phase volume was examined too, as the volume of the donor phase results in the achievement of the equilibrium state. Thus, different values of sample volume (100, 200, 500, 1000, and 1400 µL) were tested. In each case, no significant differences between the obtained peak areas were observed. Therefore, a volume of 200 µL was selected as the optimal parameter. In that volume, chemical equilibrium was established, and more compounds were not extracted to the acceptor phase.

2.1.3. Influence of the Extraction Time and Injection Parameters Additionally, the effect of extraction time was examined to improve extraction efficiency. The time of extraction can increase the analyte equilibrium between two phases: organic and aqueous. Analytes can migrate from the donor phase to an organic solvent over a period. Furthermore, excessive extraction time can cause drop instability. In contrast, a short extraction time may cause problems with achieving an equilibrium state. The three-phase SDME-CE system is a combination of three simultaneously occurring processes: extraction of the compound from an aqueous donor phase, diffusion through a hydrophobic organic thin octanol layer, and re-extraction to the aqueous acceptor phase. By adjusting the pH of the donor and acceptor phases, SDME can be carried out effectively. It is known that the form of these analytes will change with the change of solution pH and thereby affect their -solubility and extractability. The influence of extraction time was evaluated by applying different times: 1, 3, 6, 9, and 12 min. Generally, extraction times longer than 3 minutes decreased extraction efficiency, and the peak areas of analytes were smaller. Above 6 minutes of extraction, the examined compounds were not preconcentrated, and the dissolution of the drop into the donor and the acceptor phase was observed. Therefore, for further analysis, we chose 3 minutes as the extraction time. Injection time is another crucial factor in the SDME-CE procedure. Any addition of organic solvents into capillary electrophoresis can lead to problems with the current. It is essential to inject only the acceptor phase, without the organic solvent (octanol). In this case, a hydrodynamic injection was applied, and the best injection time was 1 s under a pressure of 0.1 psi. Finally, the best preconcentration effect and separation efficiency were obtained for the following conditions: donor phase pH—4, donor phase volume—200 µL, extraction time—3 min, injection—1 s at a pressure of 0.1 psi, and octanol Molecules 2020, 25, 1566 6 of 12 used as an organic phase. Figure3 shows the electropherogram obtained during the analysis of an 1 aqueous mixture of standards PSC and MUS at a concentration of 25 mg L− , with (a) and without (b) Molecules 2020, 25, x FOR PEER REVIEW 6 of 12 the preconcentration step using developed SDME in-line CE method. The presented results indicate that the analyzed compounds were sufficiently preconcentrated and separated using the developed presented results indicate that the analyzed compounds were sufficiently preconcentrated and separatedSDME-CE using system. the The developed total time SDME of analysis-CE system. was less The than total 7 minutes.time of analysis The obtained was less enrichment than 7 minutes. factors were 158 for MUS and 174 for PSC, respectively. Recovery values of over 89% for muscimol and almost The obtained enrichment factors were 158 for MUS and 174 for PSC, respectively. Recovery values of over97 % 89% for psilocinfor muscimol were obtained,and almost taking 97 % intofor psilocin consideration were obtained that the, maximumtaking into amountconsideration of enrichment that the factor for this SDME-CE working system was about 178. maximum amount of enrichment factor for this SDME-CE working system was about 178.

1 FigureFigure 3 3.. ElectropherogramsElectropherograms of of 25 25 mg L −1 aqueousaqueous samples samples of of standards standards (PSC (PSC and and MUS) MUS) during during CE CE analysisanalysis ( a)) and enriched byby 33 minmin SDME-CESDME-CE ((bb).). CECE conditions:conditions: BGE—25BGE—25 mM mM phosphate phosphate bu bufferffer pH pH 3; µ 3;SDME-CE SDME-CE conditions: conditions: Sample Sample volume: volume: 200 200L; OrganicµL; Organic phase: phase: octanol; octanol; extraction extraction time: 180 time: s; injection: 180 s; 0.1 psi, 1s; n = 6, RSD < 5.9%). injection: 0.1 psi, 1s; n = 6, RSD < 5.9%). 2.2. Real Samples Analysis 2.2. Real Samples Analysis The applicability of the developed SDME-CE method was demonstrated for the urine sample analysis.The applicability The obtained of electropherograms the developed SDME from- aCE SDME-CE method analysiswas demonstrated of a blank urinefor the sample urine (a) sample and a analysis. The obtained electropherograms from a SDME-CE analysis of a blank urine sample (a) and a sample spiked with 10 mg L−1 of the studied hallucinogenic metabolites are presented in Figure 4. This clearly shows the sample clean-up capability of SDME, as well as the sample preconcentration effect. Among many sample matrix components in urine, only a few are shown in the electropherogram. The first peak (denoted by an asterisk) is related to creatinine, identified by sample spiking. Creatinine, having pKa values of 4.9 and 9.2 and a structure similar to muscimol, was also

Molecules 2020, 25, x FOR PEER REVIEW 7 of 12

effectively extracted from the sample (donor phase) into octanol, and then re-extracted to the acceptor phase. Molecules 2020, 25, 1566 7 of 12 The obtained enrichment factors were over 120 for MUS and 160 for PSC, respectively, i.e., recovery values of over 67% for muscimol and almost 90 % for psilocin. The preconcentration effect of the analyzed spiked urine1 samples was only 10–15 % lower compared with the solution of sample spiked with 10 mg L− of the studied hallucinogenic metabolites are presented in Figure4. This clearlystandards shows prepared the sample in distilled clean-up water. capability The calibration of SDME, curves as well of asthe the studied sample analytes preconcentration in spiked urine effect. −1 Amongsamples many were sample linear matrix in the componentsrange of 0.05–50 in urine, mg L only, with a few the arecorrelation shown incoefficients the electropherogram. from 0.9911 to The 0.9992. The limit of detection in urine was 0.004 mg L−1 for PSC and 0.016 mg L−1 for MUS at a signal- first peak (denoted by an asterisk) is related to creatinine, identified by sample spiking. Creatinine, to-noise (S/N) ratio of 3. The limits of quantification varied from 0.014 mg L−1 for PSC and 0.045 mg having pKa values of 4.9 and 9.2 and a structure similar to muscimol, was also effectively extracted L−1 for MUS. The intraday and interday relative standard deviations (RSD, n = 6) of migration time (n from the sample (donor phase) into octanol, and then re-extracted to the acceptor phase. = 6) were in the range of 0.3–8.5% and 1.0–11.3%, respectively.

(a)

(b)

Figure 4. SDME-CE analysis of urine samples: electropherograms of (a) blank urine and (b) spiked 1 urine sample with PSC and MUS (10 mg L− ). SDME-CE conditions: BGE—25 mM phosphate buffer pH 3 (also acceptor phase); Sample volume: 200 µL of blank urine or spiked urine; Organic phase (single drop): octanol; Extraction time: 180 s; Injection: 0.1 psi, 1s; n = 6, RSD < 8.5%).* -creatinine. Molecules 2020, 25, 1566 8 of 12

The obtained enrichment factors were over 120 for MUS and 160 for PSC, respectively, i.e., recovery values of over 67% for muscimol and almost 90 % for psilocin. The preconcentration effect of the analyzed spiked urine samples was only 10–15 % lower compared with the solution of standards prepared in distilled water. The calibration curves of the studied analytes in spiked urine samples were Molecules 2020, 25, x FOR PEER REVIEW 8 of 12 1 linear in the range of 0.05–50 mg L− , with the correlation coefficients from 0.9911 to 0.9992. The limit 1 1 of detectionFigure 4. in SDME urine-CE was analysis 0.004 mgof urine L− forsamples: PSC andelectropherograms 0.016 mg L− forof (a MUS) blank at urine a signal-to-noise and (b) spiked (S /N) 1 1 ratio ofurine 3. Thesample limits with of PSC quantification and MUS (10 varied mg L−1). from SDME 0.014-CE mgconditions: L− for BGE PSC— and25 mM0.045 phosphate mg L− bufferfor MUS. The intradaypH 3 (also and acceptor interday phase); relative Sample standard volume: deviations 200 µL of (RSD, blank nurine= 6) or of spiked migration urine; time Organic (n = 6)phase were in the range(single of drop): 0.3–8.5% octanol; and Extraction 1.0–11.3%, time: respectively. 180 s; Injection: 0.1 psi, 1s; n = 6, RSD < 8.5%).* -creatinine.

3.3. MaterialsMaterials andand MethodsMethods 3.1. Chemicals 3.1. Chemicals Muscimol (pKa: 4.8 and 8.4 [1,43]) was purchased from Abcam (Cambridge, UK). Psilocin Muscimol (pKa: 4.8 and 8.4 [1,43]) was purchased from Abcam (Cambridge, UK). Psilocin (pKa (pKa 9.41 [4]) was from Sigma-Aldrich (Pozna´n,Poland). The chemical structures of the investigated 9.41 [4]) was from Sigma-Aldrich (Poznań, Poland). The chemical structures of the investigated analytes are shown in Figure5. analytes are shown in Figure 5.

CH HO OH 3 N CH3 N O N NH2 H MUSCIMOL PSILOCIN

FigureFigure 5. 5.Chemical Chemical structuresstructures ofof muscimolmuscimol andand psilocin.psilocin. µ AAstock stock solution solution of of the the studied studied analytes analytes was was prepared prepared in methanolin (100 (100g /µgmL)/mL and) and stored stored at 4 at◦C. 4 Sodium°C. Sodium phosphate phosphate heptahydrate, heptahydrate, toluene, dichloromethane, toluene, ethyldichloromethane, acetate, and octadecyltrimethoxysilane ethyl acetate, and (ODTS)octadecyltrimethoxysilane were obtained from Sigma-Aldrich(ODTS) were (Pozna´n, obtained Poland). from Orthophosphoric Sigma-Aldrich acid (Poznań, (85%), octanol, Poland). and sodiumOrthophosphoric hydroxide wereacid purchased(85%), octanol from, POCH.and sodium S.A. (Gliwice, hydroxide Poland). were Deionizedpurchased water from was POCH. obtained S.A. from(Gliwice, the Milli-Q Poland). system Deionized (Millipore, water MA, was USA). obtained from the Milli-Q system (Millipore, MA, USA). 3.2. SDME–CE Procedures 3.2. SDME–CE Procedures All SDME–C.E. measurements were performed on a PA 800 plus Capillary Electrophoresis System All SDME–C.E. measurements were performed on a PA 800 plus Capillary Electrophoresis from Beckmann Coulter (Brea, CA, USA) equipped with a PDA detector. All analyses were undertaken System from Beckmann Coulter (Brea, CA, USA) equipped with a PDA detector. All analyses were at a wavelength of 214 nm. An uncoated fused-silica capillary 57 cm in length (50 cm to the detector) undertaken at a wavelength of 214 nm. An uncoated fused-silica capillary 57 cm in length (50 cm to and with an inner diameter of 50 µm (Polymicro Technologies, Phoenix, AZ, USA) was used for SDME the detector) and with an inner diameter of 50 μm (Polymicro Technologies, Phoenix, AZ, USA) was analysis. This gave an acceptor phase of 1.11 µL. The capillary was conditioned daily by washing used for SDME analysis. This gave an acceptor phase of 1.11 μL. The capillary was conditioned daily it with 0.1 M NaOH (10 min), water (10 min), and BGE electrolyte (25 mM phosphate buffer pH by washing it with 0.1 M NaOH (10 min), water (10 min), and BGE electrolyte (25 mM phosphate 3, for 5 min). The voltage applied for the C.E. separation was held at 30 kV during analysis. The buffer pH 3, for 5 min). The voltage applied for the C.E. separation was held at 30 kV during analysis. temperature was maintained at 18 C. Before each run, the capillary inlet tip was washed with The temperature was maintained◦ at 18 °C. Before each run, the capillary inlet tip was washed with (3 min), immersed in a mixture of coating solution consisting of 5% ODTS in ethanol (30 s), and dried ethanol (3 min), immersed in a mixture of coating solution consisting of 5% ODTS in ethanol (30 s), in an empty vial for 5 min. This process increased the hydrophobicity of the capillary inlet tip and and dried in an empty vial for 5 min. This process increased the hydrophobicity of the capillary inlet ensured the stability of the drop suspended at the end. Next, the capillary was filled with a running tip and ensured the stability of the drop suspended at the end. Next, the capillary was filled with a buffer (acceptor phase) for 60 s (20 psi), and then octanol (organic phase) was injected into it (3 s at running buffer (acceptor phase) for 60 s (20 psi), and then octanol (organic phase) was injected into it 0.4 psi). Finally, the capillary inlet tip was transferred into a vial with a sample solution (donor phase), (3 s at 0.4 psi). Finally, the capillary inlet tip was transferred into a vial with a sample solution (donor and backpressure was applied (31 s, 0.7 psi) to form a drop of acceptor phase covered with octanol. phase), and backpressure was applied (31 s, 0.7 psi) to form a drop of acceptor phase covered with Then, the capillary outlet tip was transferred to the acceptor phase. The use of backpressure (0.7 psi) octanol. Then, the capillary outlet tip was transferred to the acceptor phase. The use of backpressure (0.7 psi) for 30 s every 60 s of the extraction allowed us to stabilize the drop size and shape. After 180 s of extraction, the enriched acceptor phase was injected into the capillary for 1 s at 0.1 psi. Next, the capillary inlet tip was placed in ethanol to remove octanol from the tip for 30 s, the capillary was placed in vials with the BGE electrolyte, and the electrophoresis process was conducted. The enrichment factor (EF) in the case of this Three-Phase SDME-CE system can be approximated as the ratio of final and initial concentrations of the analytes in the aqueous back-extractive and aqueous sample phases, respectively [30,44]. Extraction efficiency was calculated from the following equation:

Molecules 2020, 25, 1566 9 of 12 for 30 s every 60 s of the extraction allowed us to stabilize the drop size and shape. After 180 s of extraction, the enriched acceptor phase was injected into the capillary for 1 s at 0.1 psi. Next, the capillary inlet tip was placed in ethanol to remove octanol from the tip for 30 s, the capillary was placed in vials with the BGE electrolyte, and the electrophoresis process was conducted. The enrichment factor (EF) in the case of this Three-Phase SDME-CE system can be approximated as the ratio of final and initial concentrations of the analytes in the aqueous back-extractive and aqueous sample phases, Molecules 2020, 25, x FOR PEER REVIEW 9 of 12 respectively [30,44]. Extraction efficiency was calculated from the following equation: E = nA/nI, where n and n are the number of moles (analyte) input in the system during the extraction time and E = nA/nII, whereA nI and nA are the number of moles (analyte) input in the system during the extraction collectedtime and collected in the acceptor, in the acceptor, respectively. respectively. E is an analytically E is an analytically important important parameter parameter and should and should not be confusednot be confused with recovery. with recovery. It can be It measured can be measured directly (as directly E) or according (as E) or to according the alternative to the formula alternative E’ = (n nw)/n , where nw is the number of moles leaving in the donor phase after extraction. The value of formulaI − E’I = (nI − nw)/nI, where nw is the number of moles leaving in the donor phase after extraction. EThe indicates value of how E indicates much of how the inputmuch of of analytes the input arrives of analytes in the arrives acceptor, in whilethe acceptor, E’ measures while how E’ measures much is removedhow much from is removed the donor from into the the donor organic into phase. the organic The scheme phase. ofThe the scheme SDME of procedure the SDME is procedure described inis Figuredescribed6. in Figure 6.

Figure 6. Scheme of SDME SDME-CE-CE procedure.procedure. 3.3. Urine Samples Analysis 3.3. Urine Samples Analysis Blank urine samples were provided by a healthy female volunteer who did not take any medication Blank urine samples were provided by a healthy female volunteer who did not take any during the study. All urine samples were collected and stored in PTFE flasks at 4 C before use. Before medication during the study. All urine samples were collected and stored in◦ PTFE flasks at 4 °C analysis, samples were spiked with psilocin and muscimol at an appropriate concentration level, before use. Before analysis, samples were spiked with psilocin and muscimol at an appropriate diluted with water (1:1, v/v), adjusted to pH 4 (85% orthophosphoric acid), and filtered with 0.45 µm concentration level, diluted with water (1:1, v/v), adjusted to pH 4 (85% orthophosphoric acid), and PTFE filters. filtered with 0.45 µm PTFE filters. 4. Conclusions 4. Conclusions The purpose of this study was to develop a new, fast, and reliable SDME-CE method for the The purpose of this study was to develop a new, fast, and reliable SDME-CE method for the isolation, preconcentration, and determination of fungal hallucinogenic metabolites in urine samples. isolation, preconcentration, and determination of fungal hallucinogenic metabolites in urine samples. For the first time, the two different fungal derivatives, muscimol and psilocin ( and indole For the first time, the two different fungal derivatives, muscimol and psilocin (isoxazole and indole compounds) were analyzed in a biological fluid, i.e., human urine. It was demonstrated that the compounds) were analyzed in a biological fluid, i.e., human urine. It was demonstrated that the developed SDME-CE method provides not only the preconcentration of the studied analytes, but also an developed SDME-CE method provides not only the preconcentration of the studied analytes, but also efficient sample clean-up step. The time-consuming sample preparation procedure has been practically an efficient sample clean-up step. The time-consuming sample preparation procedure has been practically omitted. The obtained LOD values are sufficient for qualitative and quantitative determination, and the method can easily be applied in analyses of urine samples during routine analytical procedures.

Author Contributions: Conceptualization, A.P.; methodology, A.P. and K.Z.; validation, K.Z..; formal analysis, K.Z.; investigation, K.Z. and A.P.; writing—original draft preparation, A.P..; writing—review and editing, A.P.; supervision, P.W.; All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Wroclaw Research Centre EIT+ under the project “Biotechnologies and advanced medical technologies”–BioMed (POIG.01.01.02-02-003/08) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2)

Molecules 2020, 25, 1566 10 of 12 omitted. The obtained LOD values are sufficient for qualitative and quantitative determination, and the method can easily be applied in analyses of urine samples during routine analytical procedures.

Author Contributions: Conceptualization, A.P.; methodology, A.P. and K.Z.; validation, K.Z.; formal analysis, K.Z.; investigation, K.Z. and A.P.; writing—original draft preparation, A.P.; writing—review and editing, A.P.; supervision, P.P.W.; All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Wroclaw Research Centre EIT+ under the project “Biotechnologies and advanced medical technologies”–BioMed (POIG.01.01.02-02-003/08) financed from the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Sample Availability: Samples of the compounds are not available from the authors.

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